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Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy.

Kee AJ, Schevzov G, Nair-Shalliker V, Robinson CS, Vrhovski B, Ghoddusi M, Qiu MR, Lin JJ, Weinberger R, Gunning PW, Hardeman EC - J. Cell Biol. (2004)

Bottom Line: Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals.These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure.Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

View Article: PubMed Central - PubMed

Affiliation: Muscle Development Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia.

ABSTRACT
Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

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The NS Tms from the γ-gene and a γ-actin are colocalized to a novel Z-line adjacent region of the sarcomere. Confocal immunofluorescent images of semi-thin (0.5–1.0 μm) longitudinal sections through adult soleus muscle show that Tms detected by CG3 and γ9d (A, C, D, F, G, and I, red signal) are localized to a restricted area either side of Z-line (B, C, H, and I, delineated by the green α-actinin staining), but not including the Z-line. The restricted localization of these NS Tms to either side of the Z-line is in marked contrast to the broad region of the actin thin filament of the sarcomere (E and F, phalloidin). In longitudinal sections there appears to be little difference in the Z-line adjacent localization of the Tms detected by CG3 and γ9d (J–L). Antibodies to a γ-actin also stained the Z-line adjacent region (M–O) and this staining was coincident with the staining for both CG3 (P–R) and γ9d (S–U). In some sections, γ-actin staining was observed at the Z-line (M and O, arrowheads; R, inset). Double staining of muscle sections was performed by applying both the primary/secondary antibody pairs sequentially. Bars, 2.5 μm.
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fig4: The NS Tms from the γ-gene and a γ-actin are colocalized to a novel Z-line adjacent region of the sarcomere. Confocal immunofluorescent images of semi-thin (0.5–1.0 μm) longitudinal sections through adult soleus muscle show that Tms detected by CG3 and γ9d (A, C, D, F, G, and I, red signal) are localized to a restricted area either side of Z-line (B, C, H, and I, delineated by the green α-actinin staining), but not including the Z-line. The restricted localization of these NS Tms to either side of the Z-line is in marked contrast to the broad region of the actin thin filament of the sarcomere (E and F, phalloidin). In longitudinal sections there appears to be little difference in the Z-line adjacent localization of the Tms detected by CG3 and γ9d (J–L). Antibodies to a γ-actin also stained the Z-line adjacent region (M–O) and this staining was coincident with the staining for both CG3 (P–R) and γ9d (S–U). In some sections, γ-actin staining was observed at the Z-line (M and O, arrowheads; R, inset). Double staining of muscle sections was performed by applying both the primary/secondary antibody pairs sequentially. Bars, 2.5 μm.

Mentions: Studies in a number of nonmuscle systems have shown that the NS Tms are sorted to very specific intracellular locations (Gunning et al., 1998a,b). Therefore, we have stained longitudinal sections of muscle with the γ9d and CG3 antibodies to define the localization of Tm5NM1 and Tm5NM-34kd in muscle. Both antibodies produced strong striated staining that was distinct from the thin filament of the sarcomere. Specifically, both CG3 and γ9d (Fig. 5, A, D, and G, red staining) showed thin lines of staining adjacent to the Z-line (Fig. 5, B and H, green α-actinin staining) that did not correspond to the entire actin thin filament (Fig. 5 F, compare red CG3 and green phalloidin/filamentous-actin staining). The CG3 and γ9d staining is likely to represent the localization of Tm5NM-34kd and Tm5NM1, respectively, as on Western blots these were the predominant Tms detected by these antibodies in the soleus muscle (Fig. 2, A and B, respectively). On longitudinal sections the two Tms colocalized with each other in this Z-line adjacent region (Fig. 4 L, yellow staining in the merged image) and both colocalized in this region with the γ-actin antibody (Fig. 4, P–U). Costaining of sections with antibodies to γ-actin and the Z-line protein α-actinin indicates that γ-actin is at the Z-line adjacent region (Fig. 4 O) and also at the Z-line itself (indicated by the colocalized yellow signal of γ-actin and α-actinin in Fig. 4 O). The NS Tm banding pattern around the Z-line does not correspond to any known pattern of Tm staining in skeletal myofibers. Together, the data suggests that a γ-actin and these NS Tms are part of a novel cytoskeleton adjacent to the Z-line.


Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy.

Kee AJ, Schevzov G, Nair-Shalliker V, Robinson CS, Vrhovski B, Ghoddusi M, Qiu MR, Lin JJ, Weinberger R, Gunning PW, Hardeman EC - J. Cell Biol. (2004)

The NS Tms from the γ-gene and a γ-actin are colocalized to a novel Z-line adjacent region of the sarcomere. Confocal immunofluorescent images of semi-thin (0.5–1.0 μm) longitudinal sections through adult soleus muscle show that Tms detected by CG3 and γ9d (A, C, D, F, G, and I, red signal) are localized to a restricted area either side of Z-line (B, C, H, and I, delineated by the green α-actinin staining), but not including the Z-line. The restricted localization of these NS Tms to either side of the Z-line is in marked contrast to the broad region of the actin thin filament of the sarcomere (E and F, phalloidin). In longitudinal sections there appears to be little difference in the Z-line adjacent localization of the Tms detected by CG3 and γ9d (J–L). Antibodies to a γ-actin also stained the Z-line adjacent region (M–O) and this staining was coincident with the staining for both CG3 (P–R) and γ9d (S–U). In some sections, γ-actin staining was observed at the Z-line (M and O, arrowheads; R, inset). Double staining of muscle sections was performed by applying both the primary/secondary antibody pairs sequentially. Bars, 2.5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172434&req=5

fig4: The NS Tms from the γ-gene and a γ-actin are colocalized to a novel Z-line adjacent region of the sarcomere. Confocal immunofluorescent images of semi-thin (0.5–1.0 μm) longitudinal sections through adult soleus muscle show that Tms detected by CG3 and γ9d (A, C, D, F, G, and I, red signal) are localized to a restricted area either side of Z-line (B, C, H, and I, delineated by the green α-actinin staining), but not including the Z-line. The restricted localization of these NS Tms to either side of the Z-line is in marked contrast to the broad region of the actin thin filament of the sarcomere (E and F, phalloidin). In longitudinal sections there appears to be little difference in the Z-line adjacent localization of the Tms detected by CG3 and γ9d (J–L). Antibodies to a γ-actin also stained the Z-line adjacent region (M–O) and this staining was coincident with the staining for both CG3 (P–R) and γ9d (S–U). In some sections, γ-actin staining was observed at the Z-line (M and O, arrowheads; R, inset). Double staining of muscle sections was performed by applying both the primary/secondary antibody pairs sequentially. Bars, 2.5 μm.
Mentions: Studies in a number of nonmuscle systems have shown that the NS Tms are sorted to very specific intracellular locations (Gunning et al., 1998a,b). Therefore, we have stained longitudinal sections of muscle with the γ9d and CG3 antibodies to define the localization of Tm5NM1 and Tm5NM-34kd in muscle. Both antibodies produced strong striated staining that was distinct from the thin filament of the sarcomere. Specifically, both CG3 and γ9d (Fig. 5, A, D, and G, red staining) showed thin lines of staining adjacent to the Z-line (Fig. 5, B and H, green α-actinin staining) that did not correspond to the entire actin thin filament (Fig. 5 F, compare red CG3 and green phalloidin/filamentous-actin staining). The CG3 and γ9d staining is likely to represent the localization of Tm5NM-34kd and Tm5NM1, respectively, as on Western blots these were the predominant Tms detected by these antibodies in the soleus muscle (Fig. 2, A and B, respectively). On longitudinal sections the two Tms colocalized with each other in this Z-line adjacent region (Fig. 4 L, yellow staining in the merged image) and both colocalized in this region with the γ-actin antibody (Fig. 4, P–U). Costaining of sections with antibodies to γ-actin and the Z-line protein α-actinin indicates that γ-actin is at the Z-line adjacent region (Fig. 4 O) and also at the Z-line itself (indicated by the colocalized yellow signal of γ-actin and α-actinin in Fig. 4 O). The NS Tm banding pattern around the Z-line does not correspond to any known pattern of Tm staining in skeletal myofibers. Together, the data suggests that a γ-actin and these NS Tms are part of a novel cytoskeleton adjacent to the Z-line.

Bottom Line: Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals.These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure.Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

View Article: PubMed Central - PubMed

Affiliation: Muscle Development Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia.

ABSTRACT
Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

Show MeSH
Related in: MedlinePlus